Androgen receptor-dependent gene expression control

ABSTRACT

Disclosed is the use of at least one amine oxidase inhibitor for modulating the activity of the lysine-specific demethylase (LSD1) in a mammal and to pharmaceutical compositions for controlling the androgen receptor-dependent gene expression, comprising an effective dose of at least one amine oxidase inhibitor suitable for modulating the activity of the lysine-specific demethylase (LSD1) in a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/884,568, the entire disclosure of which is incorporated by referenceherein, which is a U.S. National Stage of International Application No.PCT/EP2006/001446, filed Feb. 16, 2006, which claims priority ofEuropean Patent Application No. 05 003 596.3, filed Feb. 18, 2005.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of at least one amine oxidaseinhibitor for modulating the activity of the lysine-specific demethylase(LSD1) in a mammal or for the manufacture of a medicament for modulatingthe activity of the lysine-specific demethylase (LSD1) in a mammal. Theinvention also relates to a pharmaceutical composition for controllingthe androgen receptor-dependent gene expression, comprising an effectivedose of at least one amine oxidase inhibitor suitable for modulating theactivity of the lysine-specific demethylase (LSD1) in a mammal. Theinvention also relates to a method for controlling the androgenreceptor-dependent gene expression in a mammal, said process comprisingthe step of administering to said mammal, on a suitable route, aneffective dose of a pharmaceutical composition modulating an activity ofthe lysine-specific demethylase (LSD1) in a mammal Finally, theinvention also relates to assay systems allowing to test LSD1 modulatorsfor their ability to modulate, preferably inhibit, LSD1 function.

2. Discussion of Background Information

The androgen receptor (AR) is a member of the steroid hormone receptorfamily of transcription factors which regulate diverse biologicalfunctions including cell growth and differentiation, development,homeostasis and various organ functions in a mammal, particularly in ahuman. By binding suitable ligands like androgens to the ligand bindingdomain, functions of the AR are activated which are essential for thedifferentiation, development and maintenance of male or femalereproductive organs and non-reproductive organs (as, for example, theprostate or the mammae).

Transcriptional regulation by nuclear receptors such as the androgenreceptor (AR) involves interaction with multiple factors that act inboth a sequential and combinatorial manner to reorganize chromatin¹.Central to this dynamic reorganization is the modification of corehistones. The N-terminal tails of histones are subject to variouscovalent modifications such as acetylation, phosphorylation,ubiquitination and methylation by specific chromatin-modifying enzymes².Histone methylation at specific lysine residues is linked to bothtranscriptional repression and activation. When searching for new ARinteracting proteins, Lysine specific demethylase 1 (LSD1)³ was found tobe one example of the chromatin-modifying enzymes.

LSD1 contains a centrally located swirm domain which functions as aputative protein-protein interaction motif, and also contains aC-terminal amine oxidase (AO) domain that harbours the demethylaseactivity³ (FIG. 1 b). Endogenous LSD1 and AR associate in vivo inandrogen-sensitive tissues such as testis (FIG. 1 a). To map theinteraction domain between LSD1 and AR in vitro, GST pull-down analyseswith labelled LSD1 and mutants thereof together with GST-AR fusionproteins were performed. As shown in FIG. 1 b, full-length LSD1, as wellas the swirm domain (LSD1 175-246) and the AO domain (LSD1 247-852)associate with either the N-terminus (NTD), the DNA binding domain(DBD), or the ligand-binding domain (LBD) of AR. In contrast, neitherthe N-terminus of LSD1 (LSD1 1-174) nor the GST control interact withAR.

It was now surprisingly found that the demethylating enzyme LSD1 isexpressed ubiquitously in human and murine fetal and adult tissues (FIG.2 a and data not shown). Furthermore, it was also detected that LSD1 isfound in the same cells (and in the same sub-cellular areas) where theAR is located (FIGS. 2 c, d). In the course of the research resultinginto the present invention, the above (and further) findings led to theconclusion that the demethylating enzyme LDS1 may exert a controllinginfluence on androgen-dependent gene expression. Furthermore, it wasfound that monoamine oxidase inhibitors as, for example pargyline,clorgyline or deprenyl (=selegiline) may be used to control demethylaseactivity and thereby regulate the AR. Thus, a specific modulation ofLSD1 activity might by a promising therapeutic target in tissues wherethe AR plays a pivotal physiological role.

SUMMARY OF THE INVENTION

Hence, the invention relates to the use of at least one amine oxidaseinhibitor for modulating the activity of the lysine-specific demethylase(LSD1) in a mammal.

Furthermore, the invention relates to the use of at least one amineoxidase inhibitor for the manufacture of a medicament for modulating theactivity of the lysine-specific demethylase (LSD1) in a mammal.

Preferred embodiments of the use-related aspect of the invention are setforth below.

Furthermore, the invention relates to a pharmaceutical composition forcontrolling the AR-dependent gene expression, comprising an effectivedose of at least one amine oxidase inhibitor suitable for modulating theactivity of the lysine-specific demethylase (LSD1) in a mammal.

Preferred embodiments of the pharmaceutical composition of the inventionare set forth below.

The invention also relates to a method for controlling the androgenreceptor-dependent gene expression in a mammal, said process comprisingthe step of administering to said mammal, on a suitable route, aneffective dose of a pharmaceutical composition modulating an activity ofthe lysine-specific demethylase (LSD1) in a mammal.

Preferred embodiments of the method of the invention are set forthbelow.

The invention also relates to assay systems allowing to test LSD1modulators for their ability to modulate LSD1 function, wherein at leastone chemical substance assumed to exert modulation of LSD1 function issubjected to one or more reaction(s) wherein said LSD1 is involved underconditions similar or identical to physiological conditions.

Preferred embodiments of the assay systems according to the inventionare set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further explained in the following description byreferring to the Figures. However, the embodiments addressed in theFigures are considered to only exemplify the invention and should not beconstrued to restrict the invention. In the Figures,

FIG. 1 shows the following: (a) LSD1 interacts with AR and LSD1 in vivoand in vitro. (b) AR co-immunoprecipitates with LSD1. Extracts frommouse testis were immunoprecipitated with an .alpha.-LSD1 antibody orcontrol rabbit IgG. Ten percent of the extract used forimmunoprecipitation was loaded as input. Western blots were decoratedwith α-AR and α-LSD1 antibodies. a, GST pull-down assays were performedwith labelled LSD1 mutants and the corresponding bacterially expressedGST-AR fusion proteins (150 mM KCl/0.15% NP40). GST proteins were usedas control. (NTD; N-terminal domain, DBD; DNA-binding domain, LBD;ligand-binding domain).

FIG. 2 shows LSD1 expression analyses. Expression of LSD1 mRNA in humantissues was examined by Northern blot analyses on a Human MultipleTissue Expression Array (a) and a Northern blot of human testis (b).(c): Immunohistochemical staining of LSD1 and AR in human normal andtumour prostate. LSD1 (A, B) and AR (C, D) immunoreactivity is detectedin the secretory epithelium of normal prostate (A, C, arrows) and tumourcells (B, D, arrows). All sections were taken from the same radicalprostatectomy specimen. Magnification: X250. (d) Sub-cellularlocalization of endogenous LSD1 and AR in the human LNCaP prostatetumour cells. AR (red) co-localises with LSD1 (green) in the nucleusupon addition of the AR agonist+R1881.

FIG. 3 shows how LSD1 interacts with chromatin: (a): Coomassie bluestaining reveals interaction of bacterially expressed GST-LSD1 with corehistones in vitro (600 mM KCl/0.3% NP40). (b): Labelled LSD1 interactswith the sepharose coupled N-terminal tail of histone H3. LNCaP cellswere incubated with or without R1881 (c, d, e), treated with or withoutpargyline (d) or transfected with siRNA (e). ChIP or Re-ChIP wereperformed with the indicated antibodies. The precipitated chromatin wasamplified by PCR using the primers flanking the promoter region (AREI+II), the middle region (middle), or the enhancer region (ARE III) ofthe AR-regulated PSA gene. siRNA mediated knockdown of LSD1 is verifiedby Western blot analysis (e, lower panel) using α-AR and α-LSD1antibodies.

FIG. 4 shows how LSD1 controls AR-induced transcriptional activity andcell proliferation. 293 (a, b, d, e), CV-1 (c), and LNCaP (f, g) cellswere transfected with the indicated AR-dependent reporters the inpresence of AR expression plasmid (a-e). Cells were treated with orwithout R1881, pargyline, deprenyl, or chlorgyline. LSD1-inducedligand-dependent activation of AR (a, b, c) is mediated by the AO domain(LSD1 247-852, d) and blocked by monoamine oxidase inhibitors (e). InLNCaP cells, pargyline (f) and siRNA-mediated LSD1 knockdown block ARactivity (g, left panel). LSD1 knockdown inhibits R1881-induced LNCaPcell proliferation (h, left panel). Knockdown of LSD1 is verified byimmunofluorescence (g, right panel, arrows) and Western blot analysis(h, right panel) using α-AR and α-LSD1 antibodies. Bars representmean+SD (>5).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention relates to the use of at least oneamine oxidase inhibitor for modulating the activity of thelysine-specific demethylase (LSD1) in a mammal.

In a second aspect, the invention relates to the use of at least oneamine oxidase inhibitor for the manufacture of a medicament formodulating the activity of the lysine-specific demethylase (LSD1) in amammal.

In accordance with the invention, at least one amine oxidase inhibitoris used. There may be used one amine oxidase inhibitor or there may beused several amine oxidase inhibitors. In preferred embodiments of theinvention, the use comprises one amine oxidase inhibitor. Any amineoxidase inhibitor may be employed in accordance with the presentinvention. However, in preferred embodiments, the at least one amineoxidase inhibitor or the one amine oxidase inhibitor is selected fromthe group of the monoamine oxidase inhibitors (MAOIs) comprising bothmonoamine oxidase A and monoamine oxydase B (MAO-A and MAO-B)inhibitors. These compounds can be nardil (phenelzine sulfate),phenelzin, parnate (tranylcypromine sulfate), tranylcypromine,isocarbazid, selegiline, deprenyl, chlorgyline, pargyline, furazolidon,marplan (isocarboxazid), 1-deprenyl (Eldepryl), moclobemide (Aurorex orManerix), furazolidone, harmine, harmaline, tetrahydroharmine,nialamide, or any extract from plant, insect, fish, mammals thatcontains MAOIs. Even more preferably, the at least one amine oxidaseinhibitor is selected from pargyline, clorgyline and deprenyl.Advantageously and, hence, most preferred, the amine oxidase inhibitoris pargyline.

According to the present invention, the at least one amine oxidaseinhibitor is used, for example is used for the manufacture of amedicament, for modulating the activity of the lysine-specificdemethylase, which is usually abbreviated as “LSD1”. The term“modulating”, as used in the present specification and claims, means achange either in the direction of improving the activity or in thedirection of reducing the activity; in accordance with the invention, ablocking of the LDS1 activity is preferred.

In a preferred embodiment of the inventive uses, the activity of LSD1modulated is the LSD1 demethylating activity. This means that the LSD1exerts an influence as a catalyst in a chemical reaction where targetmethyl groups in a polymer molecule are removed, and thereby anyinfluence on the molecule's activity is effected. To give just oneexample, an amine oxidase inhibitor used in accordance with a preferredembodiment of the invention may block demethylation of mono- anddimethyl H3-K9 during androgen-induced transcription. In a furtherpreferred embodiment of the invention, when using at least one amineoxidase inhibitor, the demethylase activity of LSD1 controlled is thedemethylating action of LSD1 on repressing histone marks on the histoneH3 and/or the histone H4, preferably on repressing histone marks on thelysine residue 9 on the histone H3 (H3-K9), and/or the lysine residue 20on the histone H4 (H4-K20), more preferably on the repressing histonemarks on mono- and dimethyl H3-K9 and/or H4-K20, thereby increasing ARregulated gene expression.

In another preferred embodiment of the invention, the mammal inconnection to which the amine oxidase inhibitors are used is a human.Even more preferred, when applying the invention to a human, the LSD1demethylase activity is targeted to tissues where the AR plays a pivotalphysiological role, preferably wherein the LSD1 demethylase activity istargeted to the brain, testis or prostate of a human, and/or any othertissue where both LSD1 and AR are co-expressed and co-localize.

In another aspect, the invention relates to a pharmaceutical compositionfor controlling the androgen receptor-dependent gene expression,comprising an effective dose of at least one amine oxidase inhibitorsuitable for modulating the activity of the lysine-specific demethylase(LSD1) in a mammal.

In accordance with the invention, it is particularly preferred that theactivity of LSD1 modulated by applying the at least one amine oxidaseinhibitor is the demethylating activity of LSD1.

In a preferred pharmaceutical composition according to the invention,one amine oxidase inhibitor is used, although the use of more than oneamine oxidase inhibitor is possible and may be advantageous. In evenmore preferred embodiments, the pharmaceutical compositions comprise atleast one, particularly preferred exactly one amine oxidase inhibitorselected from the group of the monoamine oxidase inhibitors (MAOIs)comprising both monoamine oxidase A and monoamine oxydase B (MAO-A andMAO-B) inhibitors. These compounds can be nardil (phenelzine sulfate),phenelzin, parnate (tranylcypromine sulfate), tranylcypromine,isocarbazid, selegiline, deprenyl, chlorgyline, pargyline, furazolidon,marplan (isocarboxazid), 1-deprenyl (Eldepryl), moclobemide (Aurorex orManerix), furazolidone, harmine, harmaline, tetrahydroharmine,nialamide, or any extract from plant, insect, fish, mammals thatcontains MAOIs, preferably wherein the amine oxidase inhibitors areselected from pargyline, clorgyline and deprenyl, more preferablywherein the amine oxidase inhibitor is pargyline.

It goes without saying that preferred pharmaceutical compositionsaccording to the invention, in addition to the amine oxidase inhibitor,may contain further components which a skilled person may select inaccordance with his ordinary skill. Those components may comprisesolvents, carriers, excipients, auxiliary substances by which particularproperties of the composition may be established and/or adjusted; suchsubstances may exert an own effect or may contribute to contribute toeffects exerted by other components. Examples of such additionalsubstances can be selected by a person having ordinary skill in thistechnical field in accordance with the requirements, are will known and,hence, need no further detailed description here.

The invention, in another aspect, also relates to a method forcontrolling the androgen receptor-dependent gene expression in a mammal,said process comprising the step of administering to said mammal, on asuitable route, an effective dose of a pharmaceutical compositionmodulating an activity of the lysine-specific demethylase (LSD1) in amammal.

In the method of the invention, it is preferred that the activity ofLSD1 modulated is the LSD1 demethylating activity. In preferredembodiments of the method, the modulation is effected by means of atleast one amine oxidase inhibitor. In a preferred method according tothe invention, one amine oxidase inhibitor is used, although the use ofmore than one amine oxidase inhibitor is possible and may beadvantageous. In even more preferred embodiments, the method comprisethe application or administration of at least one, particularlypreferred exactly one amine oxidase inhibitor selected from the group ofthe monoamine oxidase inhibitors (MAOIs) comprising both monoamineoxidase A and monoamine oxydase B (MAO-A and MAO-B) inhibitors. Thesecompounds can be nardil (phenelzine sulfate), phenelzin, parnate(tranylcypromine sulfate), tranylcypromine, isocarbazid, selegiline,deprenyl, chlorgyline, pargyline, furazolidon, marplan (isocarboxazid),1-deprenyl (Eldepryl), moclobemide (Aurorex or Manerix), furazolidone,harmine, harmaline, tetrahydroharmine, nialamide, or any extract fromplant, insect, fish, mammals that contains MAOIs, preferably wherein theamine oxidase inhibitors are selected from pargyline, clorgyline anddeprenyl, more preferably wherein the amine oxidase inhibitor ispargyline.

Particularly preferred embodiments of the method of the invention arecharacterized by that the demethylase activity of LSD1 controlled is thedemethylating action of LSD1 on repressing histone marks on the histoneH3 and/or the histone H4, preferably on repressing histone marks on thelysine residue 9 on the histone H3 (H3-K9), and/or the lysine residue 20on the histone H4 (H4-K20), more preferably on the repressing histonemarks on mono- and dimethyl H3-K9 and/or H4-K20, thereby increasing ARregulated gene expression.

The routes on which the administration of the effective dose of thepharmaceutical composition according to the invention to said mammal canbe performed, can be any route of administration conceivable. Theadministration route may be selected by a skilled person in accordancewith his ordinary skill and the requirements of the case. Just tomention few examples, the routes may be the oral, buccal, pulmonal,nasal, transdermal, intravenous, subcutaneous, intracutaneous,intramuscular, rectal, vaginal or intrathecal administration routes,optionally together with per se known carriers, adjuvants and additives.The oral, intraveneous, subcutaneous or intracutaneous administrationroutes are preferred.

With respect to the targets concerned, the LSD1 demethylase activity maybe directed to any target in a mammalian body, particularly in a humanbody. Preferably, the LSD1 demethylase activity is targeted to tissueswhere the AR plays a pivotal physiological role, preferably wherein theLSD1 demethylase activity is targeted to the brain, testis or prostateof a mammal, preferably of a human.

The invention also relates to an assay system allowing to test LSD1inhibitors for their ability to inhibit LSD1 function, wherein at leastone chemical substance assumed to exert inhibition of LSD1 function issubjected to one or more reaction(s) wherein said LSD1 is involved underconditions similar or identical to physiological conditions.

The term “ability to modulate LDS1 function”, as used in the presentspecification and claims, mean the ability of a chemical substanceassumed or alleged to be a modulator of the enzyme Lysine specificdemethylase (LSD1) to modulate the function of said enzyme. Themodulation may either be an initiating and/or activating modulation ormay be a decelerating or desactivating or inhibiting or even blockingmodulation. In accordance with the assay system of the presentinvention, an inhibiting or blocking modulation is preferred.

As chemical substances, basically all chemical substances may be testedby the assay system of the present invention which substances areassumed or alleged to exert a modulating action on LSD1. The reaction(s)which is/are used in the assay system of the present invention may beone reaction or may be several reactions, e. g. a reaction sequence orreaction cascade where LSD1 is involved in one or several of the stepsmaking up the reaction. In preferred embodiments of the assay systems ofthe present invention, the reaction(s) is/are (a) reaction(s) whereinLSD1 is also involved in its natural environment under naturalconditions. In other words: The assay system of the present inventionmakes use of such biochemical reactions or physiological reactionswherein LSD1 is involved when exerting its natural modulating action, e.g. on the AR.

The term “physiological conditions” as used in the present specificationand claims is understood to mean conditions which allow a mammal or,specifically, a human to exist and to be under acceptable livingconditions without any undue burden concerning temperature, pressure,acidity/basicity, humidity/aqueous conditions of liquid systems, oxygencontent of the gaseous environment etc. Such conditions are applied tothe assay system of the present invention during the test phase. Inaccordance with preferred embodiments of the present invention, saidphysiological conditions are conditions established in conducting saidassay system which are similar or identical to conditions present in oneor more than one reaction(s) where LSD1 acts under physiologicalconditions.

A preferred embodiment of the present invention relates to an assaysystem, wherein the reaction the at least one chemical substance issubjected to, with the aim of modulating, preferably blocking saidreaction, is the reaction of demethylation, by LSD1, on repressinghistone marks on specific lysine residues of said histones. Morepreferably, the reaction is the reaction of demethylation, by LSD1, onrepressing histone marks on the histone H3 or on the histone H4, morepreferably is the reaction of demethylation, by LSD1, on repressinghistone marks on the lysine residue 9 on the histone H3 (H3-K9) and/orthe lysine residue 20 on the histone H4 (H4-K20), most preferably is thereaction of demethylation, by LSD1, on the repressing histone marks onmono- and dimethyl H3-K9 and/or H4-K20.

Another preferred embodiment of the present invention relates to anassay system, wherein the reaction the at least one chemical substanceis subjected to, with the aim of modulating, preferably blocking saidreaction, is the reaction of ligand-dependent LSD1-induced superactionof AR in 293- or CV-1-cells, thus modulating, preferably blocking theligand-dependent AR activity.

A further preferred embodiment of the present invention relates to anassay system, wherein the reaction the at least one chemical substanceis subjected to, with the aim of modulating, preferably blocking saidreaction, is the reaction of androgen-dependent LNCaP- or MCF-7-cellproliferation, thus modulating, preferably blocking saidandrogen-dependent cell proliferation.

The invention is described in more detail below, without restricting itto those embodiments specifically addressed in the above description aswell as in the subsequent description of preferred embodiments.

To examine the expression pattern of LSD1, there were performed Northernblot analyses. LSD1 mRNA is ubiquitously expressed in human and murinefetal and adult tissues (FIG. 2 a and data not shown) as a transcript of3.3 kb (FIG. 2 b). To investigate LSD1 localisation in prostate, therewere used immunohistochemical analyses. As shown in FIG. 2 c, LSD1 isdetected in the epithelium of normal prostate and in tumour cells.Importantly, these cells also express AR (FIG. 2 c) indicating that LSD1and AR co-localise.

Next, the sub-cellular localisation of endogenous LSD1 and AR in humanLNCaP prostate cancer cells was studied by immunofluorescence (FIG. 2d). LSD1 is present in the nucleus in the absence and presence of the ARagonist R1881. Addition of R1881 results in nuclear co-localisation ofAR and LSD1. Taken together, the data show that LSD1 is a nuclearprotein that co-localises with AR in androgen-sensitive tissues such asprostate.

Since LSD1 was found to associate with chromatin and demethylates H3-K4in vitro³, it was examined whether LSD1 directly interacts with corehistones. Interaction analyses demonstrated physical association withcore histones in vitro (FIG. 3 a). Furthermore, the analyses show thatLSD1 interacts with the N-terminal tail of histone H3 (FIG. 3 b).

To determine whether LSD1 and AR associate with chromatin in vivo, LNCaPcells treated with R1881 were subjected to chromatin immunoprecipitation(ChIP). As shown in FIG. 3 c, genomic DNA corresponding to the androgenresponse elements (ARE I+II and ARE III) located in the promoter andenhancer of the prostate specific antigen (PSA) gene, respectively wasimmunoprecipitated in a ligand-dependent manner with α-AR antibodies.Genomic DNA derived from a region between the enhancer and promoter wasnot enriched, thus demonstrating specificity (FIG. 3 c). LSD1 associateswith chromatin both in the presence or absence of ligand (FIG. 3 c).

To demonstrate that LSD1 and AR form ligand-dependent complexes onchromatinized AREs, agonist-treated LNCaP cells were subjected tosequential chromatin immunoprecipitation (Re-ChIP), first with an α-ARantibody and next with either α-LSD1 or control α-rabbit IgG antibodies.Importantly, both ARE containing regions were enriched, demonstratingthat LSD1 and AR form a ligand-dependent complex on chromatin (FIG. 3c).

Since PSA gene expression is induced by AR in an agonist-dependentmanner the methylation levels of repressive histone marks were analysed,such as histone 3 at lysine 9 (H3-K9), histone 3 at lysine 27 (H3-K27),and histone 4 at lysine 20 (H4-K20). Androgen-induced transcription isaccompanied by a robust decrease in mono-, di-, and trimethyl H3-K9 atthe PSA promoter (FIG. 3 d).

Since LSD1 is an AO that catalyses demethylation, a test was conductedwhether monoamine oxidase inhibitors such as pargyline(N-methyl-N-2-propynyl-benzylamine), clorgyline(N-methyl-N-propargyl-3-(2,4-dichlorophenoxy-)propylamine) or deprenyl(=seregeline; (R)-(−)-N,2-dimethyl-N-2-propynylphenethylamine) mightinterfere with LSD1 function. Importantly, pargyline blocksdemethylation of mono- and dimethyl H3-K9 during androgen-inducedtranscription, whereas methylation levels of trimethyl H3-K9 and themethylation status of H3-K27 and, H4-K20 remains unchanged (FIG. 3 d anddata not shown). Interestingly, methylation of histone H3 at lysine 4(H3-K4) is not altered in vivo (data not shown).

To prove that the ligand-dependent demethylation of mono- and dimethylH3-K9 is executed by LSD1, LNCaP cells were transfected with siRNAsdirected against LSD1 or an unrelated control. This leads to efficientand specific down-regulation of endogenous LSD1, but does not affect thelevel of endogenous AR (FIG. 3 e, lower panel). Importantly, LSD1knockdown blocks ligand-dependent demethylation of mono- and dimethylH3-K9 (FIG. 3 e). The amount of total H3 is not influenced by LSD1knockdown (FIG. 3 e). Taken together, these data show theligand-dependent association of LSD1 and AR on chromatinized AREs at thepromoter of the PSA gene. This leads to the specific demethylation ofthe repressive histone marks mono- and dimethyl H3-K9.

Next, there were performed transient transfection assays to test whetherLSD1 modulates the transcriptional activity of AR. Co-expression of LSD1and AR results in a strong ligand-dependent activation of anMMTV-luciferase reporter (FIG. 4 a), which is not observed in theabsence of either ligand or AR (data not shown). Stimulation of ARactivity by LSD1 is potent in different cell lines, and bothAR-responsive minimal, synthetic and complex promoters were activated byLSD1 in a ligand-dependent manner (FIG. 4 b, c). Stimulation of ARactivity is selective since LSD1 does not affect the transcriptionalactivity of the related steroid hormone receptors (data not shown).

Furthermore, it is demonstrated that the AO domain (LSD1 247-852) ofLSD1 suffices to stimulate AR- and ligand-dependent reporter geneactivity whereas the N-terminus (LSD1 1-175) and the swirm domain (LSD1176-246) fail to do so (FIG. 4 d). All LSD1 mutants are present in thenucleus and expressed at similar levels (data not shown).

To investigate if displacement of repressive histone marks by LSD1results in increased AR regulated gene expression, monoamine oxidaseinhibitors such as pargyline, clorgyline, and deprenyl were used intransient transfections. These inhibitors severely impair LSD1-inducedactivation of AR (FIG. 4 e). Importantly, in LNCaP prostate tumourcells, which express endogenous AR, only androgen-dependent but notunrelated reporters such as TK-LUC are inhibited by pargyline, thusdemonstrating specificity (FIG. 4f).

Next, endogenously expressed LSD1 was efficiently used in LNCaP cells byvector (pSUPER-LSD1) mediated RNAi (FIG. 4 g, right panel). ParallelingLSD1 knockdown, a significant ligand-dependent decrease of PSA-LUCreporter gene expression was observed indicating impairedtranscriptional activity of AR as a consequence of LSD1 knockdown (FIG.4 g, left panel). Since LSD1 governs AR transcriptional activity otherandrogen-controlled functions such as androgen-dependent cell growthmight be regulated by LSD1.

To address this issue, LNCaP cells were infected with a lentivirus(pLV-THM-LSD1) expressing siRNA directed against LSD1. Infection withpLV-THM-LSD1 causes efficient and specific down-regulation of endogenousLSD1 but does not affect the level of endogenous AR (FIG. 4 h, rightpanel). Importantly, when compared to cells transduced with thepLV-THM-control virus, androgen-induced proliferation of LNCaP cells isdramatically inhibited by pLV-THM-LSD1 mediated LSD1 knockdown (FIG. 4h, left panel). This data demonstrate the physiological importance ofLSD1 in the control of androgen-induced gene regulation and cellproliferation.

In summary, the above data demonstrate that AR function is controlled bythe demethylase LSD1. LSD1 and AR associate at chromatinized AREs of ARtarget genes a ligand-dependent manner, which results in concomitantdemethylation of the repressive histone marks mono- and dimethyl H3-K9.LSD1 has been described as a component of co-repressor complexes⁴⁻⁷ anda recent model proposes that LSD1 represses transcription of genessilenced by Co-REST due to demethylation of the activating histone markson H3-K4³. However, when complexed with AR, LSD1 demethylates therepressing histone marks mono- and dimethyl H3-K9 and thereby promotesgene activation. Thus, depending on the specific interacting partners,LSD1 action might result in either gene silencing or activation. Ofimportance is our observation that inhibitors such as pargyline controlthe demethylase activity of LSD1 and thereby regulate AR. Thus, specificmodulation of LSD1 activity might be a promising therapeutic target intissues such as brain, testis, prostate where AR plays a pivotalphysiological role.

Methods Plasmids

The following plasmids were described previously: pSG5-AR, CMX-Flag,GST-AR-NTD, GST-AR-DBD, GST-AR-LBD, MMTV-LUC, and TK-LUC⁸;ARE_(2X)-TATA-LUC, ARE_(2X)-TK-LUC⁹; Slp-ARU-TATA-LUC¹⁰; PSA-LUC¹¹;pLV-THM (http://www.tronolab.unige.ch/); pSUPER¹². To constructCMX-Flag-LSD1, CMX-Flag-LSD1 1-174, CMX-Flag-LSD1 175-246, andCMX-Flag-LSD1 247-854 the corresponding fragments were amplified by PCRand inserted at the EcoRI/NheI site of CMX-Flag. pSUPER-control,pSUPER-LSD1 and pLV-THM-LSD1 were constructed according to(http://www.tronolab.unige.ch/) and as published¹². Sequences can beobtained upon request.

Immunohistochemistry

Polyclonal rabbit α-LSD1 antibody was generated according to standardprocedures. Stainings were performed using a protocol¹³ for antigenretrieval and indirect immunoperoxidase. α-AR 441 (Santa Cruz) andα-LSD1 were used at a dilution of 1:75 and 1:500, rabbit IgG and mouseIgG (1:500; Dako) were used as secondary antibodies and immunoreactionswere visualised with the ABC-complex diluted 1:50 in PBS (Vectastain,Vector).

Cell Culture and Transfections

293 and CV-1 cells were cultured and transfected as described¹³. LNCaPcells were cultured in phenol-red-free RPMI1640 supplemented with 10%double-stripped fetal calf serum (dsFCS) and transfected with Effectene(Qiagen). The following amounts per well were used: MMTV-LUC,ARE_(2x)-TATA-LUC, ARE_(2x)-TK-LUC, TK-LUC, PSA-LUC, Slp-ARU-TATA-LUC500 ng each, 25 ng expression plasmids for AR; 500 ng expressionplasmids for LSD1 1-174, LSD1 175-246, LSD1 247-852, pSUPER-control, andpSUPER-LSD1 100 to 500 ng expression plasmids for LSD1 were transfectedper well. Chemicals were obtained as indicated: pargyline (Sigma);deprenyl and chlorgyline (ICN Biomedicals Inc.); R1881 (Schering AG,Berlin). Cells were treated with or without 10⁻¹⁰ M R1881, 3×10⁻³ Mpargyline, 1×10⁻³ M deprenyl, or 1×10⁻⁴ M chlorgyline for 18 hours asindicated. Luciferase activity was assayed as described¹⁴. Allexperiments were repeated at least five times in duplicate.

Chromatin Immunoprecipitation

ChIP experiments were performed essentially as described¹⁵. LNCaP cellswere treated for 18 hours with or without pargyline and for 210 min withor without 10⁻⁸ M R1881 as indicated. LNCaP cells were transfected threedays before harvesting for ChIP with or without siRNA (Qiagen) followingthe manufacture's instructions. Immunoprecipitation was performed withspecific antibodies (α-monoMeK9H3, α-diMeK9H3, α-triMeK9H3,α-monoMeK4H3, α-diMeK4H3, α-triMeK4H3, α-H3 (abcam), α-LSD1, and α-ARPG21 (Upstate Biotechnology) on GammaBind™-Sepharose 4B (GE-Healthcare).For PCR, 1-5 μl out of 50 μl DNA extract was used. For Re-ChIP assays,immunoprecipitations were sequentially washed with TSE I, TSE II, bufferIII, and TE¹⁵. Complexes were eluted by incubation with 10 mM DTT at 37°C. for 30 min, diluted 50 times with dilution buffer¹⁵ followed by asecond immunoprecipitation with the indicated antibody. Primer sequenceswere as follows: middle; PSA (−2223/−1951) 5′-TGGGTTGGGTCAGGTTTTGGTT-3′and 5′-TCTTCCCCTGTTTCTAGTTGAGTG-3′; PCR primers for ARE I+II (PSA(−459/−121)) and ARE III (PSA (−4288/−3922)) have been describedpreviously¹⁶.

Immunofluorescence

Cells were analysed essentially as described¹⁴. Primary antibodystaining was performed with the indicated dilutions: α-AR 441 (1:500)and α-LSD1 (1:5000). Sub-cellular localisation was visualised usingsecondary Alexa Fluor 488- and 546-labelled antibodies (1:6000;Molecular Probes). Nuclei were stained with 1 μg ml⁻¹ DAPI (Roche).

Co-Immunoprecipitation Assays and Western Blot Analyses

Experiments were performed essentially as described⁸.Immunoprecipitations from extracts of murine testis were performed witheither α-LSD1 antibody or controlrabbit IgG. Western blots weredecorated as indicated. Ten percent of testis extract was loaded asinput.

In Vitro Pull-Down Assays

GST pull-down assays were performed with equal amounts of GST or GSTfusion proteins as described⁸ using buffer containing either 150 mM KCl,0.15% NP40 (FIG. 1 b) or 600 mM KCl, 0.3% NP40 (FIG. 3 a). Pull-downswith sepharose coupled histone H3 tail were performed as described¹⁷ in20 mM Tris pH 8.5, 150 mM NaCl, 0.5% NP40. Ten percent of the in vitrotranslated proteins were loaded as input.

mRNA Analyses

Northern blot analyses were performed with a Human Multiple TissueExpression Array and a Human Multiple Tissue Northern Blot (BDBiosciences Clontech) with an LSD1-specific probe spanning either by1-741 or by 1-2556, labelled with StripEZ (Ambion), and hybridized asrecommended.

Cell Proliferation Assay

pLV-THM-control and pLV-THM-LSD1 were used to produce recombinantlenti-viruses to infect LNCaP cells as described¹⁸. The infected cellswere cultured for 72 hours in medium supplemented with 10% dsFCS.0.3×10⁴ cells were plated in a 96-well plate with or without 10⁻⁷ MR1881. The cell proliferation Elisa BrDU Colorimetric Assay (Roche) wasperformed according to the manufacturer's instructions. The experimentswere repeated three times in quadruplet.

Assay Systems

The invention describes the following assays to test LSD1 inhibitors intheir ability to block LSD1 function and thereby AR.

-   1) The first assay to test inhibitors that block LSD1 and AR    functions can be performed as shown in FIG. 3 d. The inhibitor    blocks the demethylation of LSD1 on repressing histone marks on the    histone H3, preferably on repressing histone marks on the lysine    residue 9 on the histone H3 (H3-K9), more preferably on the    repressing histone marks on mono- and dimethyl H3-K9.-   2) The second assay to test inhibitors that block LSD1 and AR    functions can be performed as in shown in FIGS. 4 e and 4 f. An LSD1    inhibitor blocks the ligand-dependent LSD1 induced superaction of AR    in 293 or CV-1 cells. An LSD1 inhibitor blocks the ligand-dependent    AR activity.-   3) The second assay to test inhibitors that block LSD1 and AR    functions can be performed on cells that grow in an    androgen-dependent manner such as LNCaP cells or MCF-7 cells. Such    an inhibitor, similarly to siRNA mediated knockdown of LSD1 (FIG. 4    h), blocks the androgen-induced cell proliferation.

LITERATURE

-   -   1. Glass, C. K. & Rosenfeld, M. G. The coregulator exchange in        transcriptional function of nuclear receptors. Genes Dev. 14,        121-141 (2000).    -   2. Strahl, B. D. & Allis, C. D. The language of covalent histone        modifications. Nature 403, 41-45 (2000).    -   3. Shi, Y. et al. Histone Demethylation Mediated by the Nuclear        Amine Oxidase Homolog LSD1. Cell 119, 941-953 (2004).    -   4. Shi, Y. et al. Coordinated histone modifications mediated by        a CtBP co-repressor complex. Nature 422, 735-738 (2003).    -   5. Hakimi, M. A. et al. A candidate X-linked mental retardation        gene is a component of a new family of histone        deacetylase-containing complexes. J. Biol. Chem. 278, 7234-7239        (2003).    -   6. Hakimi, M. A. et al. A core-BRAF35 complex containing histone        deacetylase mediates repression of neuronal-specific genes.        Proc. Natl Acad. Sci. USA 99, 7420-7425 (2002).    -   7. Eimer, S. et al. Loss of spr-5 bypasses the requirement for        the C. elegans presenilin sel-12 by derepressing hop-1. EMBO J.        21, 5787-5796 (2002).    -   8. Metzger, E. et al. A novel inducible transactivation domain        in the androgen receptor: implications for PRK in prostate        cancer. EMBO J. 22, 270-280 (2003).    -   9. Schüle, R. et al. Functional antagonism between oncoprotein        c-Jun and the glucocorticoid receptor. Cell 62, 1217-1226        (1990).    -   10. Verrijdt, G. et al. Functional interplay between two        response elements with distinct binding characteristics dictates        androgen specificity of the mouse sex-limited protein        enhancer. J. Biol. Chem. 277, 35191-35201 (2002).    -   11. Sun, Z., Pan, J. & Balk, S. P. Androgen receptor-associated        protein complex binds upstream of the androgen-responsive        elements in the promoters of human prostate-specific antigen and        kallikrein 2 genes. Nucleic Acids Res. 25, 3318-3325 (1997).    -   12. Brummelkamp, T. R., Bernards, R. & Agami, R. A system for        stable expression of short interfering RNAs in mammalian cells.        Science 296, 550-553 (2002).    -   13. Müller, J. M. et al. FHL2, a novel tissue-specific        coactivator of the androgen receptor. EMBO J 19, 359-369 (2000).    -   14. Müller, J. M. et al. The transcriptional coactivator FHL2        transmits Rho signals from the cell membrane into the nucleus.        EMBO J. 21, 736-748 (2002).    -   15. Shang, Y., Myers, M. & Brown, M. Formation of the androgen        receptor transcription complex. Mol. Cell. 9, 601-610 (2002).    -   16. Shatkina, L. et al. The cochaperone Bag-1L enhances androgen        receptor action via interaction with the NH2-terminal region of        the receptor. Mol. Cell. Biol. 23, 7189-7197 (2003).    -   17. Schneider, R. et al. Direct binding of INHAT to H3 tails        disrupted by modifications. J. Biol. Chem. 279, 23859-23862        (2004).    -   18. Wiznerowicz, M. & Trono, D. Conditional suppression of        cellular genes: lentivirus vector-mediated drug-inducible RNA        interference. J. Virol. 77, 8957-8961 (2003).

What is claimed is:
 1. A method for controlling androgenreceptor-dependent gene expression in a mammal with prostate cancer,wherein said method comprises administering to the mammal an effectivedose of a pharmaceutical composition that comprises at least one amineoxidase inhibitor capable of modulating an activity of lysine-specificdemethylase (LSD1) and of controlling androgen receptor-dependent geneexpression in the mammal.
 2. The method of claim 1, wherein ademethylating activity of LSD1 is modulated.
 3. The method of claim 1,wherein a demethylating action of LSD1 on repressing histone marks onhistone H3 is controlled, thereby increasing androgen receptor regulatedgene expression.
 4. The method of claim 3, wherein the histone marks onhistone H3 comprise histone marks on lysine residue 9 (H3-K9).
 5. Themethod of claim 4, wherein the histone marks on histone H3 comprisehistone marks on at least one of mono- and dimethyl H3-K9.
 6. The methodof claim 1, wherein the mammal is a human.
 7. A method of treatingprostate cancer in a mammal, wherein said method comprises administeringto the mammal an effective dose of a pharmaceutical composition thatcomprises at least one amine oxidase inhibitor capable of modulating anactivity of lysine-specific demethylase (LSD1) and of controllingandrogen receptor-dependent gene expression in the mammal.
 8. The methodof claim 7, wherein a demethylating activity of LSD1 is modulated. 9.The method of claim 7, wherein a demethylating action of LSD1 onrepressing histone marks on histone H3 is controlled, thereby increasingandrogen receptor regulated gene expression.
 10. The method of claim 9,wherein the histone marks on histone H3 comprise histone marks on lysineresidue 9 (H3-K9).
 11. The method of claim 10, wherein the histone markson histone H3 comprise histone marks on at least one of mono- anddimethyl H3-K9.
 12. The method of claim 7, wherein the mammal is ahuman.